Figure 3. Mm infections are dynamic during antibiotic treatment.
(A–D) Tracking of untreated Mm infections in individual larvae. (A) Fluorescence images of a representative larva at 3 and 6 dpi. Arrows, enlarging granulomas, arrowheads, shrinking granulomas. Scale bar, 400 µm. (B) Cartoon indicating "head" region (gray) primarily containing organs, and "tail" region (stippled) comprised mostly of muscle. (C) Enumeration of expanding and contracting granulomas over time. Differential region-specific outcomes of granulomas were statistically significant for changes occurring between days 7 and 11 (P=0.0022), but not for changes occurring between days 4 and 7 (P=0.2360, Fisher’s exact test).
(D and E) Hematoxylin and eosin staining showing Mm in caseating granulomas in a 33 day-old fish that was infected at 1 dpf. Red and black arrows indicate granulomas in pronephros and liver, respectively. gl, gill; sb, swim bladder; sm, somite. Scale bar 300 µm. Higher ma gnification of granulomas in boxed inset of (D) is shown on bottom right of (E). Scale bar 50 µm.
(F–I) Larvae infected with varying Mm inocula for 4 days were then treated with 290 µM INH or left untreated for an additional 3 days. Larvae were imaged at 4 and 7 dpi (0 and 3 dpt). (F) Pre- and post-treatment log10 FPC values for individual larvae are plotted with data points from the same individual connected. (G) Raw FPC values before and after treatment, percent change, and the presence of expanding and new foci are reported for representative fish indicated in (F). (H and I) Fluorescence images of fish 11 (H) and fish 5 (I) as reported in (F and G), shown before and after treatment. Arrows, enlarging granulomas, and arrowheads, new foci. Scale bars 500 µm.
(J) A single larva was infected with 500 Mm constitutively expressing the Kaede photoactivatible GFP for 4 days. Composite red and green fluorescence images immediately after photoactivation of a granuloma (left panel) and 24 hours later (right panel). Arrows, photoactivated granuloma, arrowheads, single macrophages containing red fluorescent bacteria. Scale bar 250 µm.